Bursaphelenchus xylophilus, the pine wood nematode (PWN), is the causal agent of pine wilt disease (PWD), which causes enormous economic loss annually. According to our previous research, fomepizole, as a selective inhibitor of PWN alcohol dehydrogenase (ADH), has the potential to be a preferable lead compound for developing novel nematicides. However, the underlying molecular mechanism is still unclear. The result of molecular docking showed that the stronger interactions between fomepizole and PWN ADH at the active site of ADH were attributed to hydrogen bonds. Low-dose fomepizole had a substantial negative impact on the egg hatchability, development, oviposition, and lifespan of PWN. Transcriptome analysis indicated that 2,124 upregulated genes and 490 downregulated genes in fomepizole-treated PWN were obtained. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes indicated that fomepizole could be involved in controlling PWN vitality mainly by regulating key signaling pathways, such as the ribosome, hippo signaling pathway, and lysosome. Remarkably, the results of RNA interference indicated that the downregulated serine/threonine-protein phosphatase gene (stpp) could reduce the egg hatchability, development, oviposition, and lifespan of PWN, which was closely similar to the consequences of nematodes with low-dose fomepizole treatment. In addition, the silencing of stpp resulted in weakness of PWN pathogenicity, which indicated that stpp could be a potential drug target to control PWN.

Vancomycin and daptomycin are commonly used glycopeptide antibiotics to cure Gram-positive staphylococcal infections. The clinical isolates of mutant Staphylococcus aureus strains, Methicillin-Resistant (MRSA) and Vancomycin-Resistant (VRSA), have developed resistance against these antibiotics. A recently discovered Serine/threonine phosphatase (Stp1) is an Mn+2 containing protein at the active site with a flap sub-domain that participates in the phospho-signaling system of bacterial cell wall formation. The flap sub-domain probably regulates substrates recruitment and release with an extra Mn+2, possibly highly flexible as in the other homologous family of proteins. In this study, the flap sub-domain has been sampled with conventional and accelerated molecular dynamics (cMD and aMD) simulations to get other sub-optimal conformational states of the protein that are nearly impossible to observe through experimental methods. Trajectory analysis has shown that protein remained static in cMD while dynamic in aMD with RMSD of ∼2Å and ∼3Å, respectively. Accelerated MD has shown greater flexibility of ∼4 Å in the flap sub-domain, while cMD only captured a deviation of ∼ 2 Å. Later, the dynamic cross-correlation map (DCCM) confirmed that the flap sub-domain is significantly more flexible than the other part of the structure, indicating its role in substrate regulation. Secondary structure transition in the flap sub-domain, i.e. 3-10 helix and turn (PRO159 - ILE163) region of the flap sub-domain shifted into α-helix, which is a more stable structure. Further, the trajectory has been clustered, and conformational states extracted, which may be exploited in structure-based antibiotics discovery.Communicated by Ramaswamy H. Sarma.

Arpp19 is a potent PP2A-B55 inhibitor that regulates this phosphatase to ensure the stable phosphorylation of mitotic/meiotic substrates. At G2-M, Arpp19 is phosphorylated by the Greatwall kinase on S67. This phosphorylated Arpp19 form displays a high affinity to PP2A-B55 and a slow dephosphorylation rate, acting as a competitor of PP2A-B55 substrates. The molecular determinants conferring slow dephosphorylation kinetics to S67 are unknown. PKA also phosphorylates Arpp19. This phosphorylation performed on S109 is essential to maintain prophase I-arrest in Xenopus oocytes although the underlying signalling mechanism is elusive. Here, we characterize the molecular determinants conferring high affinity and slow dephosphorylation to S67 and controlling PP2A-B55 inhibitory activity of Arpp19. Moreover, we show that phospho-S109 restricts S67 phosphorylation by increasing its catalysis by PP2A-B55. Finally, we discover a double feed-back loop between these two phospho-sites essential to coordinate the temporal pattern of Arpp19-dependent PP2A-B55 inhibition and Cyclin B/Cdk1 activation during cell division.

Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite\'s life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca2+ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca2+ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP3R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.

The aim of our study was the identification of genetic variants associated with postoperative complications after cardiac surgery.
We conducted a prospective, double-blind, multicenter, randomized trial (RIPHeart). We performed a genome-wide association study (GWAS) in 1170 patients of both genders (871 males, 299 females) from the RIPHeart-Study cohort. Patients undergoing non-emergent cardiac surgery were included. Primary endpoint comprises a binary composite complication rate covering atrial fibrillation, delirium, non-fatal myocardial infarction, acute renal failure and/or any new stroke until hospital discharge with a maximum of fourteen days after surgery.
A total of 547,644 genotyped markers were available for analysis. Following quality control and adjustment for clinical covariate, one SNP reached genome-wide significance (PHLPP2, rs78064607, p = 3.77 × 10- 8) and 139 (adjusted for all other outcomes) SNPs showed promising association with p < 1 × 10- 5 from the GWAS.
We identified several potential loci, in particular PHLPP2, BBS9, RyR2, DUSP4 and HSPA8, associated with new-onset of atrial fibrillation, delirium, myocardial infarction, acute kidney injury and stroke after cardiac surgery.
The study was registered with ClinicalTrials.gov NCT01067703, prospectively registered on 11 Feb 2010.

Protein phosphatase 5 (PP5), mainly localized in human brain, can dephosphorylate tau protein whose high level of phosphorylation is related to Alzheimer\'s disease. Similar to other protein phosphatases, PP5 has a conserved motif in the catalytic domain that contains two binding sites for manganese (Mn2+ ) ions. Structural data indicate that two active site water molecules, one bridging the two Mn2+ ions and the other terminally coordinated with one of the Mn2+ ions (Mn1), are involved in catalysis. Recently, a density functional theory study revealed that the two water molecules can be both deprotonated to keep a neutral active site for catalysis. The theoretical study gives us an insight into the catalytic mechanism of PP5, but the knowledge of how the deprotonation states of the two water molecules affect the binding of PP5 with its substrate is still lacking. To approach this problem, molecular dynamics simulations were performed to model the four possible deprotonation states. Through structural, dynamical and energetic analyses, the results demonstrate that the deprotonation states of the two water molecules affect the structure of the active site including the distance between the two Mn2+ ions and their coordination, impact the interaction energy of residues R275, R400 and H304 which directly interact with the substrate phosphoserine, and mediate the dynamics of helix αJ which is involved in regulation of the enzyme\'s activity. Furthermore, the deprotonation state that is preferable for PP5 binding of its substrate has been identified. These findings could provide new design strategy for PP5 inhibitor.

RNA polymerase II C-terminal domain phosphatases are newly emerging family of phosphatases that contain FCPH domain with Mg＋2-binding DXDX(T/V) signature motif. Its subfamily includes small CTD phosphatases (SCPs). Recently, we identified several interacting partners of human SCP1 with appearance of dephosphorylation and O-GlcNAcylation. In this study, using an established cell line with inducible CTDSPL2 protein (a member of the new phosphatase family), proteomic screening was conducted to identify binding partners of CTDSPL2 in nuclear extract through immunoprecipitation of CTDSPL2 with its associated. This approach led to the identification of several interacting partners of CTDSPL2. This will provide a better understanding on CTDSPL2. [BMB Reports 2016; 49(6): 319-324].

Establishment of cell polarity is essential for processes such as growth and division. In fission yeast, as well as other species, polarity factors travel at the ends of microtubules to cortical sites where they associate with the membrane and subsequently maintain a polarized activity pattern despite their ability to diffuse in the membrane. In this chapter we present methods to establish an in vitro system that captures the essential features of this process. This bottom-up approach allows us to identify the minimal molecular requirements for microtubule-based cell polarity. We employ microfabrication techniques combined with surface functionalization to create rigid chambers with affinity for proteins, as well as microfluidic techniques to create and shape emulsion droplets with functionalized lipid boundaries. Preliminary results are shown demonstrating that a properly organized microtubule cytoskeleton can be confined to these confined spaces, and proteins traveling at the ends of growing microtubules can be delivered to their boundaries.

Protein phosphorylation is an important mechanism that implicates in physiology of any organism including parasitic protozoa. Metallic protein Ser/Thr protein phosphatase 5 (PP5) controls various cellular signaling pathways of Plasmodium falciparum. The structure and inhibitory mechanism of PP5 in P. falciparum is not known. In fact, no experimental structural data are available for P. falciparum Ser/Thr protein phosphatase 5 (PfPP5) till date. Hence, we have proposed computer-generated model of catalytic subunit of PfPP5 and its inhibitory mechanism was analyzed. A set of 42 known natural inhibitors of protein phosphate family were docked against metal-binding catalytic site of PfPP5 and we found that cantharidin and its derivatives shows better binding energy among them. Similarity search was performed by taking these compounds as lead compounds against PubChem and ChemBank. The search result provides 3703 similar compounds; out of which 2245 qualified the Lipinski rule of five. Further, virtual screening of these compounds was performed and selected top 25 were selected on the basis of binding energy. In continuation, rigid and flexible docking of these screened compounds was performed to get the insight of interactions. Finally, top 5 compounds were verified for ADMET properties, and then, all are subjected to MD simulations for 25 ns in order to validate their stability. Compounds CBI: 3554182, CID: 23561913, and CID: 21168680 showed most stable binding, although some of hydrogen bonds pairing varied throughout simulation. These finding would be helpful to the medicinal chemists for the development of antimalarial drugs to combat this deadly disease.

RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are a newly emerging family of phosphatases. Recently a CTD-specific phosphatase, small CTD phosphatase 1 (SCP1), has shown to act as an evolutionarily conserved transcriptional corepressor for inhibiting neuronal gene transcription in non-neuronal cells. In this study, using the established NIH/3T3 and HEK293T cells, which are expressing human SCP1 proteins under the tight control of expression by doxycycline, a proteomic screening was conducted to identify the binding partners for SCP1. Although the present findings provide the possibility for new avenues to provide to a better understanding of cellular physiology of SCP1, now these proteomic and some immunological approaches for SCP1 interactome might not represent the accurate physiological relevance in vivo. In this presentation, we focus the substrate specificity to delineate an appearance of the dephosphorylation reaction catalyzed by SCP1 phosphatase. We compared the phosphorylated sequences of the immunologically confirmed binding partners with SCP1 searched in HPRD. We found the similar sequences from CdcA3 and validated the efficiency of enzymatic catalysis for synthetic phosphopeptides the recombinant SCP1. This approach led to the identification of several interacting partners with SCP1. We suggest that CdcA3 could be an enzymatic substrate for SCP1 and that SCP1 might have the relationship with cell cycle regulation through enzymatic activity against CdcA3.